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Title

Author

Date Permissions Signed

Date of Award

2010

Document Type

Masters Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Smirnov, Serge L.

Second Advisor

Spiegel, P. Clint

Third Advisor

Anthony-Cahill, Spencer J.

Abstract

Villin is a modular protein that regulates F-actin bundles in the microvilli of absorptive epithelial cells in the intestine. At low (10-100 nM) calcium levels, Villin is an F-actin bundling agent supporting the specialized brush border membrane of the absorptive epithelium. At intermediate micromolar calcium levels, Villin nucleates and caps the barbed ends of F-actin and in high (> 100 μM) calcium Villin is an F-actin severing agent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). The amino acid sequence of Villin has seven modular domains. The first six Villin domains (D1-D6) form a "core" of ~50% sequence identity with Gelsolin; and contain a Ca2+-dependent actin-binding site associated with the D1-D3 fragment. The last domain, Villin's unique C-terminal headpiece (HP), contains the other F-actin binding site, which is Ca2+-independent (Bretsher & Weber, 1980; Glenney et al., 1980, 1981; Mooseker et al. 1980). Recent investigation by Nuclear Magnetic Resonance (NMR) Spectroscopy and Negative-Stain Electron Microscopy (EM) of the backbone dynamics and actin-binding of Villin's D6-HP, 208-residue, C-terminal modular fragment, revealed that: a) folded domains D6 and HP are interacting only via a largely unfolded 40-residue linker, and b) at millimolar calcium levels, the monomeric D6-HP fragment bundles F-actin and has two actin binding sites; one, which is previously known on HP, and the other is novel, cryptic and Ca2+-dependent, associated with domain D6 or the linker (Smirnov et al., 2007). We have investigated how the domain structure, domain-domain and linker-domain interactions in D6-HP fragment of Villin define its actin regulation properties. Toward this goal, we are: a) making the D6 and D6-HP NMR samples; b) determining the NMR resonance assignment of isolated D6; and c) elucidating the solution structure of D6 domain in isolation and within the D6-HP fragment. Our NMR data indicate that the D6 protein fragment in isolation likely adopts a Gelsolin-like fold and that HP and D6 structures in isolation resemble those in the context of the larger modular fragment D6-HP. The potential effect of the linker on the D6 and HP domains structure is exemplified by the noticeable chemical shift differences for residue 84 of D6 and residue 166 of HP (15N-HSQC spectrum of D6-HP vs. D6 and HP in isolation). These two positions are ~23 residues away from either end of the linker and located on the surface of these domains. In the absence of calcium, Gelsolin adopts a compact, inactive conformation stabilized by the 12-residue C-terminal helix. This helix was suggested to keep together Gelsolin domains D2 and D6 as a "latch" closed in low calcium and released at higher calcium levels (Robinson et al., 1999). Our ensuing structural study of D6-HP will clarify whether the linker sequence in D6-HP corresponding to this C-terminal helix of Gelsolin forms a helix as well and thus may or not undergo a gelsolin-like, calcium-induced rearrangement. The solution structure of D6 will be determined by NMR and analyzed in combination with the complete solution structure of HP and known structural properties of D6-HP. Together with the calcium and F-actin binding properties of D6 in isolation (currently under study), these data will clarify the role of the C-terminal domains of Villin in its activity as a physiologically principal actin regulator of microvilli.

Type

Text

Publisher

Western Washington University

OCLC Number

665080765

Digital Format

application/pdf

Genre/Form

Academic theses

Language

English

Rights

Copying of this thesis in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this thesis for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.